JC polyomavirus VLP (virus-like particle) with a targeting peptide

ABSTRACT

The disclosure relates to a fusion protein comprising at least a first and a second peptide, wherein —the second peptide comprises a targeting region and a first and a second interaction region, —the second peptide is located on the surface of the fusion protein; —the second peptide comprises at least two interaction pairs, wherein an interaction pair is formed by an amino acid of the first interaction region and an amino acid of the second interaction region, —the interaction between the amino acids of an interaction pair is covalent or non-covalent; and —at least one interaction pair is a covalent interaction pair in which the amino acids are covalently bound, and to virus like particles (VLP) comprising the fusion protein for use as drug delivery system. Also provided are polynucleotides encoding the fusion protein, suitable expression vectors, host cells, production methods for the fusion protein and the VLP comprising the fusion protein.

FIELD OF THE INVENTION

The present invention relates to a fusion protein comprising a peptidewith a targeting region and a first and a second interaction region fortargeting the fusion protein and to virus like particles (VLP)comprising the fusion protein for use as drug delivery system.

BACKGROUND OF THE INVENTION

In the development of specific diagnostic or therapeutic procedures, theuse of transfer systems (delivery systems) that allow a possiblecell-specific transfer of substances and nucleic acids such as, markersor agents, is of great importance. For this cell-specific transfersystems based on virus-like particles (VLP) have been developed. One ofthese systems is the VLP from the human polyomavirus John-Cunninghamvirus (JCV) as described in WO 97/19174 and EP 1270586 B1. Basis of thissystem is the ability of the VLP to package foreign cargo such as drugsor and nucleic acids instead of the viral DNA. As a VLP still has theability to specifically recognize cells and to be internalized by thecells the VLP can be used to introduce a cargo of choice into specificcells. However, the VLPs of the state art interact with a very broadspectrum of cells. For certain applications it is desirable to be ableto address only specific cell types.

In view of this prior art it is an object of the present invention toimprove VLPs for the use as a drug delivery system. In particular it isan object of the present invention to provide means to improve the cellspecificity of VLPs.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a fusion proteincomprising at least a first and a second peptide, wherein

-   -   the second peptide comprises a targeting region and a first and        a second interaction region,    -   the second peptide is located on the surface of the fusion        protein,    -   the second peptide comprises at least two interaction pairs,        wherein an interaction pair is formed by an amino acid of the        first interaction region and an amino acid of the second        interaction region,    -   the interaction region between the amino acid of an interaction        pair is covalent or non-covalent, and    -   at least one interaction pair is a covalent interaction pair in        which the amino acids are covalently bound.

The second peptide according to the invention is a modular targetingpeptide that has several advantages for targeting a protein of interestto which it is fused forming the fusion protein. The second peptideprovides a self-forming secondary structure that presents a specifictargeting sequence of choice in form of a loop spaced apart from thesurface of the fusion protein. Due to the spacing of the targetingsequence away from the surface of the fusion protein, the second peptideprovides an improved accessibility of the targeting sequence. The fusionprotein may be used in particular in combination with a virus-likeparticle (VLP) cargo transport to provide to the VLP the ability totarget specific predefined receptors/cell types.

Thus, according to the second aspect, the invention provides a VLPcomprising the fusion peptide of the first aspect of the invention.

According to a third aspect, the invention provides a pharmaceuticalcomposition comprising the VLP according to the second aspect and atleast one pharmaceutically acceptable carrier.

According to a fourth aspect, the invention provides an isolatedpolynucleotide comprising a nucleic acid sequence encoding a fusionprotein according to the first aspect of the invention. According to afifth aspect, the invention provides an expression vector comprising thenucleotide according to the fourth aspect of the invention.

According to a sixth aspect, the invention provides a host cellcomprising the expression vector according to the fifth aspect of theinvention.

According to a seventh aspect, the invention provides a process ofproducing the VLP according to the second aspect of the invention whichcomprises the steps of:

-   a) introducing a polynucleotide according to the second aspect of    the invention into a host cell;-   b) culturing the transformed host cell in a medium under conditions    leading to a protein expression with the polynucleotide as a    template,-   c) isolating the expression product from the cell and-   d) assembly of the expression product optionally with further viral    proteins into the VLP

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows two specific embodiments of the second peptide according tothe invention (a and b)

FIG. 2 provides an overview of a specific embodiment for the productionof VLPs comprising a fusion protein according to the invention.

FIG. 3 shows histograms calculated from flow cytometry data obtainedfrom MDA-MB-231 cells treated with VLPs. The histograms a) to c)correspond to the following VLPs:

-   -   a) VLP with VP1 and VP1-FITC, VLP with VP1-DE-Loop-Lyp1 and        VP1-FITC, VLP with VP1-HI-Loop-Lyp1 and VP1-FITC    -   b) VLP with VP1 and VP1-Dylight488, VLP with VP1-DE-Loop-Lyp1        and VP1-Dylight488, VLP with VP1-HI-Loop-Lyp1 and VP1-Dylight488    -   c) VLP with VP1 and VP1-Atto647, VLP with VP1-DE-Loop-Lyp1 and        VP1-Atto647, VLP with VP1-HI-Loop-Lyp1 and VP1-Atto647.    -   The X-axis represents the measured intensity of the signal, the        Y-axis the number of cells with the specific signal intensity.

FIG. 4 shows a column diagram representation of the flow cytometry dataobtained from MDA-MB-231 cells treated with VLPs. Each column representsthe percentage of cells—identified by a minimum signal strength—with apositive uptake of a specific type combination of VLP and fluorescentdye as indicated below the columns. Three different VLPs are tested:VLPs containing VP1-DE-Loop-Lyp1 or VP1-HI-Loop-Lyp1 VLPs from normalVP1. Also three different dyes are tested: FITC, Atto-488 and Atto 647.

FIG. 5 shows the detection of VLP in spheroids of a breast cancerspheroid model. The following VLPs were tested: VP1, VP1-HI-Loop-linRGD,and VP1-HI-Loop-cycRGD, each labelled with Atto-488. A mock probe wasused as a control.

FIG. 6 study design of an orthotopic breast cancer tumor model

FIG. 7 shows the flow cytometry analysis of the tumor cells from theorthotopic breast cancer tumor model of FIG. 6. Nine animals had beentreated with a VLP comprising VP1-HI-Loop-Lyp1 and three animals wereused as control.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

A “peptide” according to the present invention may be composed of anynumber of amino acids of any type, preferably naturally occurring aminoacids, which preferably are linked by peptide bonds. In particular, apeptide comprises at least 3 amino acids, preferably at least 5, atleast 7, at least 9, at least 12 or at least 15 amino acids.Furthermore, there is no upper limit for the length of a peptide.However, preferably a peptide according to the invention does not exceeda length of 500 amino acids, more preferably, it does not exceed alength of 300 amino acids; even more preferably, it is not longer than250 amino acids. Thus, the term peptide includes oligopeptides, whichusually refer to peptides with a length of 2 to 10 amino acids, andpolypeptides, which usually refer to peptides with a length of more than10 amino acids. The term “protein” refers to a peptide with at least 60,at least 80, preferably at least 100 amino acids.

The term “fusion protein” according to the invention relates to proteinsor peptides created through the joining of two or more genes thatoriginally coded for separate proteins. The genes may be naturallyoccurring from the same organism or different organisms or may besynthetic polynucleotides.

The term “exogenous” according to the invention relates to the propertyof a peptide or polynucleotide that it does not naturally occur inpolyomaviruses.

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identity”.For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:(Identical Desoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

The term “isolated” means a substance in a form or environment whichdoes not occur in nature. Non-limiting examples of isolated substancesinclude (1) any non-naturally occurring substance, (2) any substanceincluding, but not limited to, any enzyme, variant, nucleic acid,protein, peptide or cofactor, that is at least partially removed fromone or more or all of the naturally occurring constituents with which itis associated in nature.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directs theexpression of the coding sequence. Expression: The term “expression”includes any step involved in the production of the polypeptideincluding, but not limited to, transcription, post-transcriptionalmodification, translation, post-translational modification, andsecretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a polypeptide and is operablylinked to additional nucleotides that provide for its expression.

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, and the like, with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

The term “comprise”, as used herein, besides its literal meaning alsoincludes and specifically refers to the expressions “consist essentiallyof” and “consist of”. Thus, the expression “comprise” refers toembodiments wherein the subject-matter which “comprises” specificallylisted elements does not comprise further elements as well asembodiments wherein the subject-matter which “comprises” specificallylisted elements may and/or indeed does encompass further elements.Likewise, the expression “have” is to be understood as the expression“comprise”, also including and specifically referring to the expressions“consist essentially of” and “consist of”.

The term “carrier” applied to pharmaceutical compositions of theinvention refers to a diluent, excipient, or vehicle with which the VLPof the invention is administered. Such pharmaceutical carriers can besterile liquids, such as water, saline solutions, aqueous dextrosesolutions, aqueous glycerol solutions, and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical carriers are described in “Remington The Science andPractice of Pharmacy,” 21th edition, (David B. Troy ed., 2006, p.745-775, p. 802-836 and p. 837-849).

As used herein, the term “pharmaceutical composition” refers to anycomposition comprising at least the VLP with or without cargo and atleast one other ingredient, as well as any product which results,directly or indirectly, from combination, complexation, or aggregationof any two or more of the ingredients, from dissociation of one or moreof the ingredients, or from other types of reactions or interactions ofone or more of the ingredients. Accordingly, the term “pharmaceuticalcomposition” as used herein may encompass, inter alia, any compositionmade by admixing a pharmaceutically active ingredient and one or morepharmaceutically acceptable carriers.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42 degreescentigrade in 5×SSPE, 0.3 percent SDS, 200 micrograms/ml sheared anddenatured salmon sperm DNA, and 25 percent formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2 percentSDS at 50 degrees centigrade.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42 degreescentigrade in 5×SSPE, 0.3 percent SDS, 200 micrograms/ml sheared anddenatured salmon sperm DNA, and 35 percent formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at55° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42 degreescentigrade in 5×SSPE, 0.3 percent SDS, 200 micrograms/ml sheared anddenatured salmon sperm DNA, and 50 percent formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

The term “PBS” means phosphate buffered saline. It is a water-based saltsolution containing sodium phosphate, sodium chloride and, in someformulations, potassium chloride and potassium phosphate. The osmolarityand ion concentrations of the solutions match those of the human body.

2. Fusion Protein

According to a first aspect, the invention provides a fusion proteincomprising at least a first and a second peptide, wherein:

-   -   the second peptide comprises a targeting region and a first and        a second interaction region,    -   the second peptide is located on the surface of the fusion        protein,    -   the second peptide comprises at least two interaction pairs,        wherein an interaction pair is formed by an amino acid of the        first interaction region and an amino acid of the second        interaction region,    -   the interaction region between the amino acid of an interaction        pair is covalent or non-covalent, and    -   at least one interaction pair is a covalent interaction pair in        which the amino acids are covalently bound.

The second peptide according to the invention, i.e. the targetingpeptide, is based on a particular secondary structure resembling a hairpin known from single-stranded polynucleotides especially in RNAmolecules. When folded into its secondary structure, the peptidepreferably comprises two paired regions of the amino acid sequence, thefirst and second interaction region and an unpaired loop comprising thetargeting region as shown schematically in FIGS. 1a and b.

The targeting region according to the invention comprises an amino acidsequence—the targeting sequence—that is known to interact with a targetof interest, in particular a cellular receptor. The secondary structureof the second peptide may also be described as a stem loop comprising astem region and a loop region. Accordingly, the two interaction regionsof the peptide preferably form the stem and the targeting region formsthe loop (see FIGS. 1a and b ). When located on the surface of thefusion protein, the stem, i.e. the first and second interaction regionof the second peptide, lead to a sufficient spacing between the surfaceof the protein and the targeting region so that an interaction with atargeting recognizing means, in particular a cellular receptor, ispossible without steric hindrance.

The folding of the structure is based on the following theoreticprinciple. During protein folding, the amino acids on the first andsecond interaction region get into proximity.

When two complementary amino acids of the two interaction regions get inproximity to each other, they will transiently bind to each other, i.e.interact non-covalently, and, thus, form a non-covalent interactionpair. The more interaction pairs are formed at a time, the higher is thebinding strength and the longer the transient interaction of the twointeraction regions. The interaction pairs of the second peptide arepreferably set up such that the formation of these non-covalentinteraction pairs brings the amino acids of the covalent interactionpair, in particular cysteines, into proximity to each other for asufficient time so as to allow formation of a covalent bond, e.g. adisulfide-bridge. The formation of the covalent interaction pair leadsto a further stabilization of the interaction of the first and secondinteraction region of the second peptide. Accordingly, the finalsecondary structure of the second peptide with a loop including thetargeting region and a stem formed by the first and second interactionregion is formed. The loop can be regarded as a circular peptideconnected by a covalent interaction pair. It was shown for a variety ofsignaling/targeting peptides that a circular shape of the peptideimproves its recognition by the specific receptor.

Thus, according to one embodiment of the first aspect of the invention,the amino acid sequence of the targeting region is located between theamino acid sequences of the first and second interaction region. Alocation of the amino acid sequence of the targeting region between thefirst and second interaction region is required to obtain a targetingregion that is located in the loop of the folded second peptide.

The amino acid sequence of the targeting region may overlap with theamino acid sequences of the first and/or second interaction region. Inparticular, the amino acids forming the covalent interaction pair may bepart of the targeting region.

The amino acid sequence of the targeting region may be any sequence thatis recognized or binds to a target molecule, in particular a cellularreceptor. Non-limiting examples of such peptides are Lyp-1 (SEQ ID NO:1), RGD (SEQ ID NO: 60, RGD), RGR, HER2 binding peptide (SEQ ID NO: 2),CREKA peptide (SEQ ID NO: 3), NGR peptide, CPP-2 (SEQ ID NO: 4), CPP-44(SEQ ID NO: 5), F3 (SEQ ID NO: 6), RMS-P3 (SEQ ID NO: 7), F56 (SEQ IDNO: 8), LTVSPWY-peptide (SEQ ID NO: 9), WNLPWYYSVSPT-peptide (SEQ ID NO:10), SP5-2 (SEQ ID NO: 11), heparan sulfate targeting peptide (SEQ IDNO: 61, CKNEKKNKIERNNKLKQPP), CGKRK-peptide (SEQ ID NO: 62, CGKRK),CSRPRRSEC-peptide (SEQ ID NO: 63, CSRPRRSEC), CREAGRKAC-peptide (SEQ IDNO: 64, CREAGRKAC), CAGRRSAYC-peptide (SEQ ID NO: 65, CAGRRSAYC), RMS-P3(SEQ ID NO: 66, CMGTINTRTKKC), CKAAKN-peptide (SEQ ID NO: 67, CKAAKN),CSNRDARRC-peptide (SEQ ID NO: 68, CSNRDARRC), CGNSNPKSC-peptide (SEQ IDNO: 69, CGNSNPKSC), CSRESPHPC-peptide (SEQ ID NO: 70, CSRESPHPC),ASGALSPSRLDT-peptide (SEQ ID NO: 71, ASGALSPSRLDT), IL-4-receptorbinding peptide (SEQ ID NO: 72, CRKRLDRNC), and PSP1 (SEQ ID NO: 73,CLSYYPSYC).

Thus, according to one embodiment of the first aspect of the invention,the targeting region comprises a sequence selected from the groupconsisting of SEQ ID NO: 1, RGD, RGR, SEQ ID NO: 2, SEQ ID NO: 3, NGRpeptide, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 60, SEQ IDNO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73. Preferably the aminoacid sequence of the targeting region comprises SEQ ID NO: 1. In anotherpreferred embodiment, the amino acid sequence of the targeting regioncomprises SEQ ID NO: 60.

Lyp-1 is a tumor homing peptide that selectively binds thetumor-associated lymphatic vessels and tumor cells in certain tumors.The nine amino acid long peptide specifically recognizes the receptorP32. The RGD-peptide and NGR-peptide are tri-peptides composed ofL-arginine-glycine-L-aspartic acid and L-asparagine-glycine-L-arginine,respectively. The sequences are common elements in cellular recognition.RGD peptides are implicated in cellular attachment via integrins. TheHER2 binding peptide specifically targets the Human Epidermal GrowthReceptor 2 (HER2). The CREKA peptide is a tumor homing peptideidentified in phage display libraries consisting of the sequenceCys-Arg-Glu-Lys-Ala (see Simberg D, et al. Biomimetic amplification ofnanoparticle homing to tumors. Proc Natl Acad Sci USA. 2007 Jan. 16;104(3):932-6). CPP-2 and CPP-44 are tumor homing peptides described inKondo et al. Tumourlineage-homing cell-penetrating peptides asanticancer molecular delivery systems. Nat Commun. 2012 Jul. 17; 3:951.F3 comprises amino acid sequences 17-48 of High Mobility GroupNucleosomal Binding Protein 2 (HMGN2) and was identified in a phagedisplay cDNA library screen for peptides capable of homing to tumors,especially to their vascular endothelium (see (see Christian et al.,Nucleolin expressed at the cell surface is a marker of endothelial cellsin angiogenic blood vessels. J Cell Biol. 2003 Nov. 24; 163(4):871-8).RMS-P3 is a furin targeted peptide suitable for targetingRhabdomyosarcoma (RMS) cells (see Hajdin K, et al. Furin targeted drugdelivery for treatment of rhabdomyosarcoma in a mouse model. PLoS One.2010 May 3; 5(5)). F56 specifically binds to VEGF receptor Fit-1 (seeHerringson and Altin, Effective tumor targeting and enhanced anti-tumoreffect of liposomes engrafted with peptides specific for tumorlymphatics and vasculature. Int J Pharm. 2011 Jun. 15; 411(1-2):206-14).LTVSPWY-peptide and WNLPWYYSVSPT-peptide specifically bind to breastcancer cells (see Shadidi and Sioud, Identification of novel carrierpeptides for the specific delivery of therapeutics into cancer cells,FASEB J. 2003 February; 17(2):256-8). SP5-2 specifically binds tonon-small cell lung cancer (see Chang D K, et al. A Novel PeptideEnhances Therapeutic Efficacy of Liposomal Anti-Cancer Drugs in MiceModels of HumanLung Cancer, PLoS ONE 2009 (1):e4171).

According to one embodiment of the first aspect of the invention, theloop between the first and second interaction region which comprises thetargeting region has a number of amino acids in the range from 3 to 50amino acids. The number of amino acids of the loop is counted from thecovalent interaction pair “closing” the loop and consequently includesthe amino acids of the covalent interaction pair. Accordingly a numberof loop amino acids of 2 only includes the covalent interaction pair.Thus, the minimal number of amino acids in the loop is 3. The maximumlength of the loop is in principle limited by the influence of thepeptide on the folding of the fusion protein and the tendency foraggregation with higher length. Thus, the maximum number of amino acidsin the loop is preferably 25, more preferably 20, most preferably 15amino acids. According to a particularly preferred embodiment, thenumber of amino acids in the loop is in the range from 5 to 15 aminoacids.

The covalent interaction pair may be formed by any two amino acids, theside chains of which may form a covalent bond. These may be inparticular cysteines or seleno cysteines which form disulfide bridges.According to one embodiment of the first aspect of the invention, thecovalent interaction pair is formed by a cysteine in the firstinteraction region and by a cysteine in the second interaction region. Afusion protein according to the invention may comprise more than onecovalent interaction pair. For example, the fusion protein according tothe invention may comprise 7 or less, 6 or less, 5, or less, 4 or less,3 or less, 2 or less interaction pairs. The covalent interaction pairsmay be located in sequence or spaced apart. Preferably, the fusionprotein according to the invention comprises one covalent interactionpair.

According to one embodiment of the first aspect of the invention, atleast two interaction pairs are non-covalent interaction pairs in whichthe amino acids interact non-covalently. Preferably, the second peptidecomprises at least 3 non-covalent interaction pairs, more preferably atleast 4 non-covalent interaction pairs. In principal, the higher thenumber of interaction pairs, the stronger the interaction of the firstand second interaction region of the second peptide. The number ofnon-covalent interaction pairs also depends on the type of interactionof the amino acids. The non-covalent interaction may be by hydrogenbridges, van der Waal forces, hydrophobic interactions or acid-baseinteractions.

Preferably, at least a part of the non-covalent interaction pairs areacid-base interaction pairs formed by an acidic amino acid in oneinteraction region and a basic amino acid in the other interactionregion. At a neutral pH, these amino acids are charged negatively andpositively, respectively. The contrary charges of the amino acids leadto an attraction of these amino acids and consequently of theinteraction regions. Moreover, the contrary charges provide a tightbinding of binding.

According to one embodiment, the second peptide comprises 2 to 20acid-base interaction pairs, preferably 2 to 10 acid-base interactionpairs, more preferably 2 to 6 acid-base interaction pairs, mostpreferably 3 to 5 acid-base interaction pairs. The higher the number ofacid-base interaction pairs, the higher the attraction of the first andsecond interaction region. However, a number more than 20 acid-baseinteraction pairs will be problematic for the folding of the fusionprotein. A number of more than 10 acid-base interaction pairs renderscloning more problematic as very long primers have to be used. Moreover,it is assumed that the use of more than 6 acid-base interaction pairsdoes not further significantly increase the interaction of the first andsecond interaction region. With regard to ease of cloning and optimalstrength of interaction of the non-covalent interaction pairs, a numberof 3 to 5 acid-base interaction pairs are preferred. In a particularlypreferred embodiment of the first aspect of the invention the secondpeptide comprises 4 acid-base interaction pairs.

Basic amino acids according to the invention can be arginine, lysine orhistidine. Acidic amino acids according to the invention can be glutamicacid or aspartic acid.

The first and second interaction region may comprise both acidic andbasic amino acids, only acidic amino acids or only basic amino acids.The basic and acid amino acids in one interaction region may bealternating or form clusters. Non-limiting examples of alternatingsequences are: EERR (SEQ ID NO: 45), ERER (SEQ ID NO: 46), EERREE (SEQID NO: 47), RREERR (SEQ ID NO: 48). Examples of clusters are EEERRR (SEQID NO: 49), DDERKK (SEQ ID NO: 50), DDDRR (SEQ ID NO: 51). However, itis preferred that one of the interaction regions comprises a majority ofacidic amino acids and the other, consequently, a majority of basicamino acids. For example, the first inter action region comprises themainly basic sequence RRRRE (SEQ ID NO: 52) and the second interactionregion comprises the mainly acidic sequence EEEER (SEQ ID NO: 53).

According to a preferred embodiment of the first aspect of theinvention, the non-covalent interaction pairs are acid-base interactionpairs and formed by an acidic amino acid in the first interaction regionand basic amino acid in the second region. The first interaction regionmay comprise at least 2, at least 3, at least 4, at least 5, at least 6acidic amino acids. Also, the second interaction region may comprise atleast 2, at least 3, at least 4, at least 5, at least 6 basic aminoacids. Preferably, the first interaction region comprises at least 4acidic amino acids and the second interaction region comprises at least4 basic amino acids. More preferably, the first interaction regioncomprises at least 4 consecutive acidic amino acids and the secondinteraction region comprises at least 4 consecutive basic amino acids.

According to one embodiment of the first aspect of the invention, themajority of the basic amino acids are arginine. In an alternativeembodiment, the majority of the basic amino acids are lysines.Preferably, all basic amino acids in the interaction region arearginines.

The charged amino acids in the interaction regions, i.e. the acidic andbasic amino acids of the first and second interaction region may bedirectly in sequence or contain non-charged amino acids as spacers inbetween. Accordingly, two charged amino acids in the interaction regionmay be directly connected or may be separated by one or more non-chargedamino acids. The non-charged amino acids as spacer between the chargedamino acids are preferably selected from the group consisting of serineand glycine. The number of non-charged amino acids, i.e. the spacing,between two charged amino acids may be for example 0, 1, 2, 3 or 4.According to one embodiment of the invention, the spacing of the chargedamino acids within the amino acid sequence of the first interactionregion is 0 or 1. According to one embodiment of the invention thespacing of the charged amino acids in the second interaction region is 0or 1. According to one embodiment the spacing of the charged amino acidsin the first and/or second interaction region is preferably 0. Thus, thecharged amino acids in the first and/or second interaction region aredirectly connected.

In a preferred embodiment, the first interaction region comprises fourconsecutive arginines. According to one embodiment of the first aspectof the invention, the majority of the acidic amino acids are glutamicacids. According to an alternative embodiment, the majority of acidicamino acids are aspartic acids. Preferably, all acidic amino acids inthe second peptide are glutamic acids. In particular the firstinteraction region comprises the sequence EEEE (SEQ ID NO: 54) and thesecond interaction region comprises the sequence RRRR (SEQ ID NO: 55).In a further preferred embodiment, the fusion protein comprises a firstinteraction region comprising the sequence RRRRSGC (SEQ ID NO: 74) and asecond interaction region comprising the sequence CSGEEEE (SEQ ID NO:75) as depicted in FIG. 1A. In a further preferred embodiment, thefusion protein comprises a first interaction region comprising thesequence RRRRC (SEQ ID NO: 76) and a second interaction regioncomprising the sequence CEEEE (SEQ ID NO: 77) as depicted in FIG. 1B.

According to one embodiment the first interaction comprises the sequenceEGEGEGE (SEQ ID NO: 56) and the second interaction region comprises thesequence RGRGRGR (SEQ ID NO: 57). According to the one embodiment thefirst interaction comprises the sequence ESESESE (SEQ ID NO: 58) and thesecond interaction region comprises the sequence RSRSRSR (SEQ ID NO:59).

The spacing region between the covalent interaction pair or pairs andthe non-covalent interaction pair or pairs has an influence on theformation of the hair pin-like structure of the second peptide. If thenumber of amino acids forming the spacer is too high, the effect ofbringing the amino acids of the covalent interaction pair proximity bymeans of the binding of the one or more non-covalent interaction pairsmay be lost. In contrast, a too short distance may be problematic forsteric reasons. For example, the size of the side chains of the acidicand basic amino acids is bigger than the size of the side chain ofcysteines. Accordingly, if the cysteines are directly adjacent to thecharged amino acids in the second peptide a disulfide bridge might notform. Thus, according to one embodiment of the first aspect of theinvention, the number of amino acids in the first and second interactionregion between the at least one covalent interaction pair and theclosest non-covalent interaction pair is in the range from 1 to 6,preferably 1 to 4, more preferably 1 to 3. Most preferably, the spacersin both interaction regions between the at least one covalentinteraction pair and the closest non-covalent interaction pair is 2amino acids.

In addition, the type of amino acids forming the spacer between the atleast one covalent interaction pair and the closest non-covalentinteraction pair influences the formation of the covalent bond. For thespacers, polar uncharged amino acids, with short side chains arepreferred such as glycine, serine or alanine. More preferably the aminoacids of the spacers between the at least one covalent interaction pairand the closest non-covalent interaction pair are glycine and serine.According to a particularly preferred embodiment of the invention, thespacers between the covalent interaction pair and the non-covalentinteraction pair consist of one glycine and one serine.

According to one embodiment of the fusion protein the first interactionregion is Lyp-1 interaction region 1 as defined by SEQ ID NO: 20.According to one embodiment of the fusion protein the second interactionregion is Lyp-1 interaction region 2 as defined by SEQ ID NO: 21.According to one embodiment the amino acid sequence of the firstinteraction region is defined as Lyp-1 interaction region 1 and theamino acid sequence of the second interaction region is defined as Lyp-1interaction region 2.

Preferably, the second peptide is introduced into a region of the firstpeptide that is not essential for folding so that the second peptidedoes not interfere with the folding of the first peptide. Moreover, itis preferred that the second peptide is introduced into a region of thefirst peptide that is located on the surface of the first peptide whenfolded. The skilled person knows how to determine suitable positionswithin an amino acid sequence. Suitable positions are preferablydetermined from crystal structures of the protein or related proteins.Preferably, the second peptide is located in a loop of the first peptideof the fusion protein. More preferably in a loop on the surface of thefirst peptide.

The second peptide is particularly useful as a targeting peptide fortargeting virus-like particles (VLPs). VLPs may comprise cargo that isuseful only in specific cell types or toxic and must therefore addressonly specific cell types. Accordingly, it is preferred that the firstpeptide is a protein forming the capsid of a VLP.

According to a preferred embodiment, the first peptide is a polyomavirus VP1. “VP1” or “virus protein 1” according to the invention refersto a protein which is identical to or derived from the natural VP1 ofthe JC virus, having the amino acid sequence according to SEQ ID NO: 12.A protein derived from the natural VP1 of the JC virus preferably has anamino acid sequence homology or identity with the amino acid sequenceaccording to SEQ ID NO: 12 of at least 80%, of at least 85%, of at least90%, of at least 95%, of at least 97%, of at least 98%, or of at least99%, or with a sequence of at least 100 contiguous amino acids,preferably of at least 150, of at least 200, of at least 250, of atleast 300 contiguous amino acids. Most preferably, the amino acidsequence homology or identity is calculated over the entire length ofthe natural JCV-VP1. The terms “VP1 derived from the natural VP1 of theJC virus” and “VP1 derived from JC virus” in particular also includeVP1, which is identical to the natural VP1 of the JC virus. The term“VP” according to the invention also encompasses fractions andderivatives of the natural VP1, which are capable of assembling intoVLP. Preferably, said fractions and derivatives of VP1 at least compriseamino acids 32 to 316 of the amino acid sequence according to SEQ ID NO:9 or a derivative thereof. Having a homology or identity with the aminoacid sequence from amino acid position 32:316 of SEQ ID NO: 9 of atleast 80%, of at last 85%, of at least 90%, of at least 95%, of at least97%, of at least 98%, or of at least 99%.

Preferably, the first peptide is a VP1 from JCV. According to X-raycrystallography analysis the folded VP1 (e.g. PDB entry 3NXD) containsthree loops on the surface of the protein. Two of these loops, theDE-loop (aa 120-137) and the HI-loop (aa 262-272) are known to beeligible for the introduction of exogenous peptide structures as anexogenous structure introduced into the loop is accessible and ingeneral does not interfere with folding of the VP1 protein. Thus,according to one embodiment, the second peptide is located in theDE-loop or the HI-loop of VP1. Preferably, the peptide is locatedbetween amino acid 120 and 137 (DE-loop) or 262 and 272 (HI-loop) ofVP1. More preferably between amino acid 129 and 132 (DE-loop) or 265 and268 (HI-loop) of VP1. In particular, the second peptide substitutesamino acid 267 of the HI-loop. As described below, different expressionsystems are suitable for the expression of the protein. According to apreferred embodiment, the fusion protein is expressed in E. coli.

According to one embodiment of the fusion protein the first peptide is aVP1 from JCV and the second peptide comprises the Lyp-1 peptide.According to one embodiment the first peptide is a VP1 from JCV and thesecond peptide comprises the amino acid sequence with a sequencehomology or identity with the amino acid sequence according to SEQ IDNO: 13 of at least 95%. According to one embodiment the first peptide isa VP1 from JCV and the second peptide comprises a amino acid sequenceaccording to SEQ ID NO: 13. According to one embodiment the firstpeptide is a VP1 from JCV, the second peptide comprises a amino acidsequence according to SEQ ID NO: 13 and the second peptide is integratedinto the DE-loop of the VP1. According to one embodiment the firstpeptide is a VP1 from JCV, the second peptide comprises an amino acidsequence according to SEQ ID NO: 13 and the second peptide is integratedinto the HI-loop of the VP1. The fusion protein may have an amino acidsequence with a sequence homology or identity with the amino acidsequence according to SEQ ID NO: 14 of at least 95%. Alternatively, thefusion protein may have an amino acid sequence with a sequence homologyor identity with the amino acid sequence according to SEQ ID NO: 15 ofat least 95%.

3. Virus-Like Particle

According to a second aspect, the invention provides a virus-likeparticle (VLP) which comprises at least one fusion protein according toa first aspect of the invention. Preferably, the VLP is a polyoma virusVLP and the first peptide of the fusion protein is a polyoma virus VP1.

Non-limiting examples for viruses of the polyoma family are:B-lymphotropic polyomavirus (formerly known as African green monkeypolyomavirus, AGMPyV) (LPyV), Baboon polyomavirus 1 (SA12), Batpolyomavirus (formerly known as Myotis polyomavirus, MyPyV; BatPyV) BKpolyomavirus (BKPyV), Bornean orang-utan polyomavirus (OraPyV1), Bovinepolyomavirus (BPyV), California sea lion polyomavirus (SLPyV), Hamsterpolyomavirus (HaPyV), JC polyomavirus (JCPyV), Merkel Cell polyomavirus(MCPyV), Murine pneumotropic virus (formerly known as Kilham strain ofpolyomavirus, Kilham virus, K virus; MPtV), Murine polyomavirus (MPyV),Simian virus 40 (formerly known as Simian vacuolating virus 40; SV40),Squirrel monkey polyomavirus (SqPyV), Sumatran orang-utan polyomavirus(OraPyV2), Trichodysplasia spinuolsa-associated polyomavirus (TSPyV),Human polyomavirus 6 (HPyV6), Human polyomavirus 7 (HPyV7), KIpolyomavirus (formerly known as Karolinska Institute polyomavirus,KIPyV), WU polyomavirus (formerly known as Washington Universitypolyomavirus, (WUPyV), Avian polyomavirus (formerly known as BudgerigarFledgling disease polyomavirus, BFPyV, APyV), Canary polyomavirus(CaPyV), Crow polyomavirus (CPyV), Finch polyomavirus (FPyV), GooseHemorrhagic polyomavirus (GHPyV), Athymic rat polyomavirus (RatPyV),Baboon polyomavirus 2 (BPyV2), Cynomolgus polyomavirus (CyPV), Gorillagorilla gorilla polyomavirus 1 (GggPyV1), Human polyomavirus 9 (HPyV9),Trichodysplasia spinulosa-associated polyomavirus (TSV) Mastomyspolyomavirus (multimammate mouse—Mastomys species), Pan troglodytesverus polyomavirus 1a (PtvPyV1a), Pan troglodytes verus polyomavirus 2c(PtvPyV2c), Rabbit kidney vacuolating virus (RKV).

Preferably the VLP is derived from a human polyoma virus comprisingHuman polyomavirus 6 (HPyV6), Human polyomavirus 7 (HPyV7), Humanpolyomavirus 9 (HPyV9), BK polyomavirus (BKPyV), JC polyomavirus(JCPyV), Merkel Cell polyomavirus (MCPyV), KI polyomavirus (formerlyknown as Karolinska Institute polyomavirus, KIPyV), WU polyomavirus(formerly known as Washington University polyomavirus, (WUPyV),Trichodysplasia spinulosa-associated polyomavirus (TSV), human polyomavirus 10 (HPyV10), MW polyomavirus and MX polyomavirus. In a morepreferred embodiment of the invention, the VLP is derived from the humanpolyoma virus JCV.

VLPs are multi-protein structures that mimic the organization andconformation of authentic native viruses but lack the viral genome.

The virus-like particle according to the invention is preferably derivedfrom human polyomavirus. In the context of the invention, the term “fromhuman polyomavirus” refers to a VLP with structural proteins that can beisolated or extracted from polyomaviruses or which can be generated byrecombinant expression of a polyoma structural protein or a modifiedform of said structural protein.

The capsids of all polyomaviruses have a similar structural set-upincluding the proteins VP1, VP2, VP3, and agnoprotein. The icosahedralvirus capsid is formed by 72 VP1 pentamers. In the center of each of thepentamers, facing to the inside of the capsid, a VP2 or VP3 protein islocated. VP3 is identical to the C-terminal two-thirds of VP2. Thisshared region comprises inter alia the nuclear localization signal(NLS), the DNA-binding domain (DBD), and the VP1 interacting domain(VID).

“VP2” or “virus protein 2” according to the invention refers to aprotein which is identical to or derived from the natural VP2 of the JCvirus, having the amino acid sequence according to SEQ ID NO: 22. Aprotein derived from the natural VP2 of the JC virus preferably has anamino acid sequence homology or identity with the amino acid sequenceaccording to SEQ ID NO: 22 of at least 80%, of at least 85%, of at least90%, of at least 95%, of at least 97%, of at least 98%, or of at least99%, or with a sequence of at least 100 contiguous amino acids,preferably of at least 150, of at least 200, of at least 250, of atleast 300 contiguous amino acids. Most preferably, the amino acidsequence homology or identity is calculated over the entire length ofthe natural JCV-VP2.

“VP3” or “virus protein 3” according to the invention refers to aprotein which is identical to or derived from the natural VP3 of the JCvirus, having the amino acid sequence according to SEQ ID NO: 23. Aprotein derived from the natural VP3 of the JC virus preferably has anamino acid sequence homology or identity with the amino acid sequenceaccording to SEQ ID NO: 23 of at least 80%, of at least 85%, of at least90%, of at least 95%, of at least 97%, of at least 98%, or of at least99%, or with a sequence of at least 100 contiguous amino acids,preferably of at least 150, of at least 200, of at least 250, of atleast 300 contiguous amino acids. Most preferably, the amino acidsequence homology or identity is calculated over the entire length ofthe natural JCV-VP3.

With the integration of a fusion protein according to the first aspectof the invention, the VLP may be targeted to a cell of interest.Polyomavirus VLPs are formed up to 72 pentamers. Each of these pentamersconsists of 5 copies of VP1. Preferably, at least one pentamer of theicosahedral VLP capsid is formed by 1 to 5 copies of the fusion proteinof the first aspect of the invention. Pentamers of the VLP may comprise1, 2, 3, 4 or 5 copies of the fusion protein according to the firstaspect. As the pentamers form upon expression and are stable also upondisassembly of the VLP, recombinant expression of only the fusionprotein in a host cell generally leads to the formation of pentamerswith 5 copies of the fusion protein.

According to one embodiment of the second aspect of the invention theratio of pentamers formed by the fusion protein and pentamers formed byother VP1 is at least 1:71, at least 1:35, at least 1:23, at least 1:17,at least 1:11, at least 1:8, at least 1:7, at least 1:5, at least 1:3 atleast 1:2. In this regard other VP1 proteins are VP1 constructs that donot include a fusion protein according to the invention. Preferably theratio is at least 1:5. The higher the number of the fusion proteinaccording to the invention in a VLP the higher the chance that the VLPwill interact with a cell type specific for the targeting region of thefusion protein. However, the integration of a peptide into the structureof the VP1 may interfere with assembly of the VLP. Thus, it is preferredthat at least a fraction of the pentamers is formed by VP1 without apeptide insertion, preferably by a VP1 with a sequence identity of atleast 90%, of at least 95%, of at least 97%, of at least 98%, or of atleast 99% to SEQ ID NO: 9.

The VLP according to the second aspect of the invention may comprisemore than one type fusion protein according to the first aspect of theinvention. For example, the VLP may comprise one type of fusion proteinwith a targeting region specific for a first target and a second type offusion protein with a targeting region specific for a second target.Such a VLP is able to target to different targets. The two targets mayfor example be epitopes located on different cell types. Accordingly, atleast two different types of cells can be targeted. Alternatively, thetwo targets may be located on one type of cells. In this scenario thetwo targeting regions increase the chance of affecting the specific celltype by the VLP. The VLP with more than one fusion protein according tothe first aspect of the invention may comprise a first fusion proteinwith the Lyp1-peptide as targeting region and a second fusion proteinwith the LTVSPWY-peptide as targeting region. The VLP with more than onefusion protein according to the first aspect of the invention mayfurther comprise a first fusion protein with the Lyp1-peptide astargeting region and a second fusion protein with the F3-peptide astargeting region. This VLP is particularly useful for targeting breasttumor cells. Moreover, the VLP with more than one fusion proteinaccording to the first aspect of the invention may comprise a firstfusion protein with the SP5-2-peptide as targeting region and a secondfusion protein with the F3-peptide as targeting region.

The VLP according to the second aspect may further comprise minor capsidproteins such as VP2, VP3 or agno protein. The VLP preferably comprisesa fusion protein comprising a VP1 binding protein, which preferablycomprises the VP1 interacting domain of VP2 and an exogenous peptide,preferably selected from a cargo binding peptide (CBP) and endosomaltranslocating peptide (ETP).

According to one embodiment of the second aspect, the VLP comprises asecond fusion protein comprising a VP1 binding protein and an exogenouspeptide, wherein the exogenous peptide comprises a cargo-loading peptideand/or an endosomal translocating peptide (ETP).

The term “VP1 binding protein” refers to any peptide that has theability to bind to the major capsid protein VP1 of a polyomavirus. Inparticular, the VP1 binding protein is a peptide comprising the VP1interacting domain (VID) of a polyomavirus VP2/VP3 protein.

The VID may be derived from a VP2 or VP3 or differently termedfunctional equivalent thereof from any known polyomavirus.

The VP1 binding protein according to the invention comprises the VP1interacting domain of VP2 and allows a positioning of the fusion proteinand, in particular, the exogenous peptide within a VLP derived from apolyomavirus. Preferably, the VP1 interacting domain has an identity ofat least 90%, preferably at least 95%, more preferably at least 98% toSEQ ID NO: 24. Thus, the VP1 binding protein preferably comprises atleast a sequence with an identity of at least 90%, preferably at least95%, more preferably at least 98% to SEQ ID NO: 24.

The VP1 binding protein is preferably a full length polyomavirus VP2 orVP3. These proteins are naturally adapted for the interaction with theVP1. Accordingly, in one embodiment of the first aspect of theinvention, the VP1 binding protein comprises an amino acid sequence thathas an identity of at least 80%, preferably at least 90%, morepreferably at least 95% to SEQ ID NO: 22 or SEQ ID NO: 23.

However, any fragment or sub-structure of VP2 or VP3 may be sufficientfor a tight interaction with VP1 as long as it contains a functional VP1interacting domain of VP2/VP3. For example, the VP1 binding proteinaccording to the invention may include or exclude the DNA-bindingdomain. For example, the VP1 binding protein may comprise the VID andthe NLS of VP2. Further examples are a VP1 binding protein comprisingthe VID and the NLS of VP2, a VP1 binding protein comprising the VID andthe DBD of VP2, and VP1 binding protein comprising the VID, DBD and theNLS of VP2.

The VP1 binding protein may be a modified version of VP2 or VP3, e.g.mutated by insertion, deletion, or amino-acid replacement with respectto SEQ ID NO: 22 or 23. However, the VP1 binding protein may only bemodified to the point that the VP1 interacting domain is stillfunctional, i.e. still binds to a polyomavirus VP1.

The exogenous peptide may be located at any position of the secondfusion protein, i.e. at the C-terminus, at the N-terminus, or at anyposition within the amino acid sequence of the fusion protein. Thelocation of the exogenous peptide is preferably on the surface of thefolded protein. The exogenous peptide is further preferably freelyaccessible when the second fusion protein is bound to the VLP capsid.The skilled person knows how to determine positions within the aminoacid sequences that fulfill these prerequisites. The structurepredictions of VP2 or VP3 show that the N-terminus and the C-terminusare located on the surface of VP2 and VP3, and oriented to the inside ofthe polyoma virus when VP2 or VP3 is bound to a VP1 pentamer.

In one embodiment of the second aspect of the invention the exogenouspeptide forms the C-terminus or the N-terminus of the second fusionprotein. A second fusion protein containing the exogenous peptide on theC-terminus or N-terminus of the protein has the further advantage of aneasier construction of the polynucleotide encoding the fusion protein.The C-terminus is particularly preferred as the location for theexogenous peptide because it is the part of the protein that is the lastto be translated. Thus, an exogenous peptide on the C-terminus has thelowest influence on protein folding. According to one embodiment of thefirst aspect, the endosomal translocating peptide, preferably the CPP,is located on the C-terminus of the protein. Alternatively, theendosomal translocating peptide, preferably the CPP, forms theN-terminus of the second fusion protein. According to an alternativeembodiment, the cargo loading peptide, in particular the cargo bindingpeptide, forms the C-terminus of the second fusion protein.Alternatively, the cargo binding peptide may form the C-terminus.

The exogenous peptide preferably has a percentage of basic amino acidsof at least 25%, more preferably of at least 30%.

According to one embodiment of the first aspect of the invention, theexogenous peptide comprises a cargo loading peptide. A cargo loadingpeptide according to the invention is a peptide that affects thepackaging of cargo in a VLP such that the cargo is better protected fromthe surrounding of the VLP in particular in the blood plasma or inside acell.

Preferably the cargo loading peptide is a cargo-binding peptide.Depending on the application of the VLP it may be used for transportingdifferent types of cargo. Examples of cargo are single- ordouble-stranded DNA, single- or double-stranded RNA, peptides, hormones,lipids, carbohydrates, or other small organic compounds. Furtherexamples of cargos are chemotherapeutics such as alkylating agents (e.g. cyclophosphamide, calicheamicin), antimetabolites (e. g.5-fluorouracil, methotrexate), anthracyclines (e. g. doxorubicin,epirubicin), RNA polymerase inhibitors (e. g. alpha-amanitin), orcytoskeletal drugs (e. g. colchicine, cytochalasin, demecolcine,latrunculin, jasplakinolide, nocodazol, taxanes, phalloidin, swinholide,vinca alkaloids). A preferred chemotherapeutic is the taxane Paclitaxel(Taxol). Chemotherapeutics may also be small organic compounds.Accordingly, the cargo-binding peptide may be specific for one or moreof these possible cargos. A preferred cargo-binding peptide is aDNA-binding peptide. A further preferred cargo-binding peptide is anRNA-binding peptide.

As shown in the examples, a cargo-loading peptide fused to the VP2 orVP3 protein leads to an improved protection, i.e. less degradation, ofDNA packaged into VLPs. Without being bound to theory, one explanationfor this better protection of the DNA cargo is a tighter packaging ofthe VLP due to the improved interaction with the cargo. Accordingly, thecargo-loading peptide is in particular a cargo binding peptide. Wildtypepolyomavirus VP2/VP3 already contain a DNA-binding domain located at theC-terminus. However, the addition of protamine-1 to the C-terminus VP2or VP3 leads to an improved protection of the cargo with respect to thewild type VP2 or VP3.

The length of the cargo-binding peptide is in principle only limited bythe requirement that it does not interfere with the folding of thefusion protein. However, the cargo-binding peptide preferably has alength in the range from 5 to 100 amino acids, more preferably in therange from 10 to 70 amino acids, most preferably in the range from 10 to60 amino acids. In one embodiment, the length is in the range from 15 to25 amino acids.

According to one embodiment of the second aspect of the invention, theamino acid sequence of the cargo-binding peptide has a percentage ofbasic amino acids of at least 40%. Basic amino acids are positivelycharged. The positive charge facilitates a binding to negatively chargedcargo, e.g. nucleotides. Preferably, the majority of the basic aminoacids of the cargo-binding peptide are arginine residues. Accordingly,the percentage of arginine residues in the sequence of the cargo-bindingpeptide is at least 20%, more preferably at least 25%, most preferablyat least 35%. In a particularly preferred embodiment, the percentage ofarginine is at least 40%.

According to one embodiment of the cargo-binding peptide comprises astructural motif (R)_(n) wherein n is an integer of at least 2, at least3, at least 4, at least 5.

Cargo-binding peptides according to the invention may be DNA-bindingpeptides, RNA-binding peptides, peptide-binding peptides, lipid-bindingpeptides, carbohydrate-binding peptides. Examples of such peptides areprotamine-1 (PRM1), Snap tag, SAMp73 Preferably, the CBP in the fusionprotein according to the invention is protamine-1. Besides cargo-bindingpeptides the group of cargo-loading peptides also includes for exampleGFP or EGFP. According to one embodiment of the fusion protein accordingto the first aspect of the invention the cargo-loading peptide isneither GFP nor EGFP.

According to a further embodiment of the second aspect of the inventionthe amino acid sequence of the cargo-loading peptide has an identity ofat least 80%, preferably of at least 90%, more preferably of at least95% to SEQ ID NO: 25 (Protamine-1) or SEQ ID NO: 26(Protamine-1aa8-29).A sequence identity to SEQ ID NO: 25 is particularly preferred. As shownin the examples, protamine-1 bound to either VP2 or VP3 has a strongeffect on the protection of the cargo transported by VLPs.

According to an alternative embodiment of the second aspect of theinvention the exogenous peptide comprises an endosomal translocatingpeptide (ETP).

An “ETP” according to the invention is a peptide that has the ability totranslocate itself and any cargo bound to it through the endosomalmembrane. Preferred endosomal translocating peptides are in particularcell-penetrating peptides (CPP). Cell-penetrating peptides (CPPs) areshort peptides that facilitate cellular uptake of various molecularcargo (from nano-size particles to small chemical molecules or largefragments of DNA). The cargo is associated with the peptides eitherthrough chemical linkage via covalent bonds or non-covalentinteractions. The functions of the CPPs are to deliver the cargo intothe cells. A process that commonly occurs through endocytosis with thecargo delivered to the endosomes of the living mammalian cells. However,other peptides not classified as CPPs have the same function providingmeans to translocate through the endosomal membrane. Such peptides arealso included in the definition of ETPs. One example for such a peptideis a polyhistidine peptide. A polyhistidine peptide consists of at leastsix histidine (His) residues. It was shown that a polyhistidine peptidealso has a destabilizing effect on membranes. According to oneembodiment of the first aspect of the invention the ETP is not aHis₆-tag.

CPPs typically have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine, or has sequences that contain alternating pattern ofcharged amino acids and non-polar/hydrophobic amino acids. These twotypes of structures are referred to as polycationic or amphiphatic,respectively. A third class of CPPs are hydrophobic peptides, containingonly apolar residues with a low net charge or have hydrophobic aminoacid groups that are crucial for cellular uptake.

The mechanism by which the CPPs translocate the plasma membrane andfacility the delivery of molecular cargo to the cytoplasm or anorganelle is not entirely understood. However, the theories of CPPtranslocation can be classified into three main entry mechanisms: directpenetration in the membrane, endocytosis-mediated entry, andtranslocation through the formation of a transitory structure.

The CPP is in theory not limited in length; however, the peptide mustallow a correct folding of the fusion protein. The cargo-binding peptidepreferably has a length in the range of 5 to 100 amino acids, morepreferably in the range from 10 to 30 amino acids, most preferably inthe range from 15 to 25 amino acids.

Preferably, the CPPs contain in addition to the basic amino acids alsonon-polar amino acids. In particular, the CPP has a percentage ofnon-polar amino acids of at least 25%, preferably of at least 30%, morepreferably of at least 35%. The groups of CPPs differ in their relativepercentage of basic non-polar amino acids.

The first type of CPPs, the amphipathic CPPs consist of alternatingbasic and non-polar amino acids. The amphipathic form often generates apore or channel through the membrane bilayer. Examples of amphipathicCPPs are the trans-activating transcriptional activator (TAT) from humanimmunodeficiency virus-1 (HIV-1) and penetratin, a peptide derived fromthe DNA-binding domain of antennapedia homeo protein. The second type ofCPPs, the so-called polycationic CPPs include the HPV peptide L2. TheseCPPs comprise at least one cluster of basic amino acids adjacent to atleast one cluster of hydrophobic amino acids. Both regions are requiredfor full activity of the peptide. Without being bound to theory,scientific results suggest that the positive charge of the basic aminoacid cluster mediates tight association with negatively charged lipidsof the membranes and that subsequent insertion of the hydrophobic dusterinto membranes induces a torsional stress which results in membranedisruption.

Amphipathic CPPs in particular have a percentage of basic amino acids inthe range from 40 to 60%, and a percentage of non-polar amino acids inthe range from 28 to 39%. The amphipathic CPPs preferably have apercentage of arginines in the range from 18 to 36%, and a percentage oflysines in the range from 22 to 28%.

The polycationic CPPs preferably have a percentage of arginines in therange from 26 to 30%, and a percentage of lysines in the range from 3 to8%.

According to one embodiment of the first aspect of the invention, theamino acid sequence of the CPP comprises a structural motif (R)_(n),wherein _(n) is an integer of at least two, preferably of at leastthree, more preferably of at least four, and the sequence furthercomprises two or more adjacent non-polar amino acids. Polycationic CPP,such as HPV 33-L2, may have a sequence of four arginines and a sequenceof three non-polar amino acids.

Preferred CPPs according to the invention are TAT, penetratin, and HPV33-L2. TAT has an amino acid sequence as defined by SEQ ID NO: 27.Penetratin has an amino acid sequence as defined by SEQ ID NO: 28, andHPV 33-L2 has an amino acid sequence as defined by SEQ ID NO: 29. Afurther preferred CPP is a variant of HPV 33-L2, which is identified asHPV 33-L2-DD447 (SEQ ID NO: 30), differs from HPV 33-L2 by a replacementof the N-terminal phenylalanine and isoleucine by two aspartates. Thisvariant was shown to have a stronger cell-penetrating effect (Kemper etal., 2006). Further examples of CPPs according to the invention areSynB1 (SEQ ID NO: 31), SynB3 (SEQ ID NO: 32), PTD-4 (SEQ ID NO: 33),PTD-5 (SEQ ID NO: 34), FHV Coat-(35-49) (SEQ ID NO: 35), BMV Gag-(7-25)(SEQ ID NO: 36), HTLV-II Rex-(4-16) (SEQ ID NO: 37), D-Tat (SEQ ID NO:38), R9-Tat (SEQ ID NO: 39).

Thus, according to one embodiment of the first aspect of the invention,the amino acid sequence of the CPP has an identity of at least 80%,preferably of at least 90%, more preferably of at least 95%, mostpreferably of at least 98% to SEQ ID NO: 27. Alternatively, the aminoacid sequence of the CPP has an identity of at least 80%, preferably ofat least 90%, more preferably of at least 95%, most preferably of atleast 98% to SEQ ID NO: 28. The sequence of the CPP may also have anidentity of at least 80%, preferably of at least 90%, more preferably ofat least 95%, most preferably of at least 98% to SEQ ID NO: 29.Moreover, the amino acid sequence of the CPP may have an identity of atleast 80%, preferably of at least 90%, more preferably of at least 95%,most preferably of at least 98% to SEQ ID NO: 30.

According to one embodiment of the first aspect of the invention, thefusion protein comprises at least one exogenous cargo-binding peptide,and at least one exogenous CPP. That way, the fusion protein may provideto a VLP, which is used as a transport system for a specific cargo intoa cell, a tighter packaging, an improved protection of the cargo, and,in addition, an improved ability to leave the endosomal pathway andarrive at the cytoplasm. Consequently, the combination of an ETP, inparticular a CPP, and a cargo-loading peptide, in particular acargo-binding peptide together in one fusion protein with the ability tobind to the major structural protein VP-1 strongly increases the yieldof cargo entering the cytoplasm of a cell and thus increases theefficiency of a VLP mediated transport into a cell.

In a fusion protein comprising both a cargo-binding peptide and a CPP,the two peptides may be located at opposite termini of the fusionprotein. Thus, either the cargo-binding peptide forms the N-terminus ofthe fusion protein, and the CPP forms the C-terminus of the fusionprotein, or the cargo-binding peptide forms the C-terminus of the fusionprotein and the CPP forms the N-terminus of the fusion protein. Bothtermini of VP2 and VP3 are presented to the surface of the protein andto the inside of the VLP when the VP2/VP3 is attached to VP1. VP2 andVP3 both contain a DNA-binding sequence on the C-terminus of thepeptide. Thus, preferably, the exogenous cargo-binding peptide forms theC-terminus of the fusion protein and the CPP forms the N-terminus of thefusion protein.

According to one embodiment of the second aspect of the invention, oneexogenous peptide comprises the CPP and the cargo-binding peptide andforms the N- or C-terminus of the protein. A localization of the twopeptides on the C-terminus is preferred. The localization on theC-terminus is advantageous in cases in which the exogenous peptide mayinterfere with the protein folding. As protein folding already occursco-translationally, there will be less interference by an exogenouspeptide bound to the C-terminus which is only translated in the end.

It is preferred that the first peptide of the fusion protein comprisesVP1 from the same polyomavirus as the further VP1 proteins forming theVLP.

According to one embodiment of the second aspect of the invention, theVLP comprises no cargo. In the context of this invention, a VLP withoutcargo is also referred to as “empty VLP”. Preferably, the empty VLPcomprises a second fusion protein according to the invention wherein theVP1 binding protein is VP2.

Surprisingly, it was found that an empty VLP can be designed thatexhibits cytotoxic properties when introduced into a cell. An example ofa cytotoxic empty VLP is VLP comprising a second fusion proteinaccording to the invention wherein the VP1 binding protein is VP2. Dueto the cytotoxic effect this empty VLP may for example be used aschemotherapeutic agents. The cytotoxic effect is preferably cellspecific. A high cell specificity of the cytotoxic effect has theadvantage that only specific cell types can be targeted in order not toharm any healthy cells. Thus, an empty VLP preferably comprisestargeting means for specific cells on which the cytotoxic effect shouldbe acted on. This targeting means is provided by a fusion proteinaccording to the first aspect.

According to an alternative embodiment of the second aspect of theinvention, the VLP comprises a cargo. Non limiting examples of cargo aresingle-stranded or double-stranded DNA, single-stranded ordouble-stranded RNA, peptides, hormones, lipids, carbohydrates, or othersmall organic compounds or mixtures thereof. Further examples of cargosare chemotherapeutics, such as alkylating agents (e. g.cyclophosphamide, calicheamicin), antimetabolites (e. g. 5-fluorouracil,methotrexate), anthracyclines (e. g. doxorubicin, epirubicin), RNApolymerase inhibitors (e. g. alpha-amanitin), or cytoskeletal drugs (e.g. colchicine, cytochalasin, demecolcine, latrunculin, jasplakinolide,nocodazol, taxanes, phalloidin, swinholide, vinca alkaloids). Apreferred chemotherapeutic is the taxane Paditaxel (Taxol).Chemotherapeutics may also be small organic compounds. Preferably, thecargo is a substance that produces an effect in a eukaryotic cell, inparticular a mammalian cell. A preferred type of cargo is a substancethat is pharmaceutically active. Preferably, the cargo is a moleculeable to act on RNA. More preferably, the cargo is siRNA. In anotherpreferred embodiment, the cargo is a chemotherapeutic.

According to one embodiment of the second aspect of the invention theVLP is for use as a medicament. In particular, the VLP is for use in thetreatment of tumor diseases. In this regard the VLP provides a vehiclefor the cargo, preferably a pharmaceutically active ingredient, to enterthe cells of an organism. Thus, according to one embodiment the VLP isfor use as a drug delivery system. For this purpose the VLP is loaded bya drug of interest. Loading drugs into VLP is especially useful fordrugs which are too toxic to be delivered and/or too hydrophilic toenter cells on their own, for example chemotherapeutics. The loading ofthe drug is in particular performed by disassembly of the VLP intopentamers and reassembly in the presence of the cargo. The VLP drugdelivery system is then used to deliver the loaded drug to a specifictarget.

In one embodiment according to the second aspect of the invention, thecargo is a pharmaceutically active substance that is not applicable byitself to a patient or leads to strong side effects. An example of agroup of such substances is chemotherapeutic substances. According to afurther embodiment, the cargo is a diagnostic agent. Preferably, thediagnostic agent is a substance used in imaging methods. Morepreferably, the diagnostic agent is a dye, in particular a fluorescentdye. Thus, according to one embodiment of the second aspect the VLP isused as a diagnostic method. Preferably the diagnostic method includesthe visualization of metastases and/or tumors, in particular for thepreparation of a surgery.

The treatment or diagnosis of a patient with a VLP according to thesecond aspect of the invention comprises the transfer of the cargo intoa cell of an organism. Preferably, the organism is a mammal, morepreferably, a human.

In one specific embodiment the VLP according to the invention areadministered to the object in need thereof, in particular to humans,intravenously.

4. Pharmaceutical Composition

According to a third aspect, the invention provides a pharmaceuticalcomposition that comprises at least one VLP according to the secondaspect of the invention, and at least one pharmaceutically acceptableexcipient.

5. Polynucleotide

According to a fourth aspect, the invention provides an isolatedpolynucleotide that comprises a nucleic acid sequence encoding a fusionprotein according to the first aspect of the invention.

The techniques used to isolate or clone a polynucleotide encoding apeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well-known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used.

The isolated polynucleotide preferably comprises a first part encodingfirst peptide and second part encoding the second peptide. The firstpart of the polynucleotide encoding the first peptide preferably has adegree of sequence identity to the JCV-VP1 coding sequence SEQ ID NO: 7of at least 80 percent, at least 85 percent, at least 90 percent, atleast 95 percent, at least 96 percent, at least 97 percent, at least 98percent, at least 99 percent, or 100 percent. In one embodiment of thefourth aspect of the invention the first part of the polynucleotideencoding the VP1 binding protein hybridizes under very low stringencyconditions, low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with SEQ ID NO: 7.

According to one embodiment of the fourth aspect of the invention, thesecond part of the polynucleotide encoding the second peptide preferablyhas a degree of sequence identity to SEQ ID NO: 8 of at least 80percent, at least 85 percent, at least 90 percent, at least 95 percent,at least 96 percent, at least 97 percent, at least 98 percent, at least99 percent, or 100 percent, which encode a polypeptide having proteaseactivity. Preferably, the second part of the polynucleotide encoding thesecond peptide preferably hybridizes under very low stringencyconditions, low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with SEQ ID NO: 8.

6. Expression Vector

In a fifth aspect the invention also relates to expression vectorscomprising a polynucleotide according to the fourth aspect of theinvention. The expression vector further preferably comprises controlelements such as a promoter, and transcriptional and translational stopsignals. The polynucleotide of according to the fourth aspect and of thecontrol elements may be joined together to produce a recombinantexpression vector that may include one or more restriction sites toallow for insertion or substitution of the polynucleotide encoding thepolypeptide at such sites. The polynucleotide may be inserted into anappropriate expression vector for expression. In creating the expressionvector, the coding sequence is located in the expression vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide of the fourthaspect of the invention. The choice of the expression vector willtypically depend on the compatibility of the expression vector with thehost cell into which the expression vector is to be introduced. Theexpression vectors may be a linear or closed circular plasmid.

The expression vector may be adapted for cell-based or cell-freeexpression. The expression vector may be an autonomously replicatingvector, i.e., a vector that exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, an extrachromosomal element, a minichromosome, or an artificialchromosome. For autonomous replication, the vector may further comprisean origin of replication enabling the vector to replicate autonomouslyin the host cell in question. The origin of replication may be anyplasmid replicator mediating autonomous replication that functions in acell. The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. For integration intothe host cell genome, the expression vector may rely on any otherelement of the expression vector for integration into the genome byhomologous or non-homologous recombination. Alternatively, the vectormay contain additional polynucleotides for directing integration byhomologous recombination into the genome of the host cell at a preciselocation in the chromosome.

The vectors of the present invention preferably contain one or more(e.g., several) selectable markers that permit easy selection oftransformed, transfected, transduced, or the like cells. A selectablemarker is a gene the product of which provides for biocide or viralresistance, resistance to heavy metals, prototrophy to auxotrophs, andthe like.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

7. Host Cells

According to a sixth aspect the invention provides a host cell,comprising the expression vector according the fifth aspect of theinvention. The expression vector according to the fifth aspect isintroduced into a host cell so that the expression vector is maintainedas a chromosomal integrant or as a self-replicating extra-chromosomalvector as described earlier. The term “host cell” encompasses anyprogeny of a parent cell that is not identical to the parent cell due tomutations that occur during replication. The choice of a host cell willto a large extent depend upon the gene encoding the polypeptide and itssource.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.Preferably the host cell is E. coli.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell. Preferably, the host cell is an insect cell,still more preferably a lepidopteran cell, and most preferably a cellselected from the group consisting of Sf9, Sf21, Express SF+, andBTITn-5B1-4 (“TN High Five”).

8. Production Method

According to a seventh aspect the invention provides a process ofproducing the VLP according the second aspect of the invention.

The process at least comprises the steps of protein expression of thefusion protein according to the first aspect of the invention with apolynucleotide according to the fourth aspect of the invention as atemplate, purifying the fusion protein and assembling several copies ofthe fusion protein together with several copies of VP1 to form a VLP.

For expression of fusion protein cell-based or cell-free (in vitro)expression systems may be used. Common cell based systems are bacteria,such as E. coli, B. subtilis, yeast, such as S. cerevisiae or eukaryoticcell lines, such as baculovirus infected Sf9 cells mammalian cells likeCHO or HeLa.

Cell-free (In vitro) protein expression is the production of recombinantproteins in solution using biomolecular translation machinery extractedfrom cells. Cell-free protein production can be accomplished withseveral kinds and species of cell extract. Extracts used for cell-freeprotein expression are made from systems known to support high levelprotein synthesis. For example cell-free extracts capable are made fromE. coli, rabbit reticulocyte lysates (RRL), wheat germ extracts, orinsects cell (such as SF9 or SF21) lysates.

Preferably, a cell based expression system is used. Thus, according toone embodiment of the sixth aspect of the invention the process ofproducing the VLP according the second aspect of the invention comprisesat least the steps of:

-   a) introducing a polynucleotide according to the fourth aspect into    a host cell;-   b) culturing the transformed host cell in a medium under conditions    leading to a protein expression with the nucleic acid as a template;-   c) isolating the expression product from the cell; and-   d) assembly of the expression product optionally with further viral    proteins into the VLP.

Suitable host cells and expression vectors are described above. The useof baculo viruses together with insect cells, in particular SF9 cells,is preferred.

Methods of cultivation of host cells in a nutrient medium suitable forproduction of the fusion protein are well known in the art. For example,the host cell may be cultivated by shake flask cultivation, small-scaleor large-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentersperformed in a suitable medium and under conditions allowing the fusionprotein to be expressed. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection).

Depending on the host/vector system used the fusion protein may or maynot be secreted into the nutrient medium. In case it is secreted thepolypeptide can be recovered directly from the medium. Otherwise thecells are separated from the culture medium and lysed.

Methods of cell lysis are known in the art. Non-limiting examples of themethods of cell lysis are mechanical disruption, liquid homogenization,sonication, freeze-thaw procedure or mortar and pestle.

The fusion protein may be recovered using methods known in the art. Forexample, the fusion protein may be recovered from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The fusion protein may be purified by a variety of procedures known inthe art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

Expressed VP1 assembles into VLPs upon expression. The VLP may bedisassembled by incubation with a reducing agent and a chelating agent(e.g. 5 mM EDTA). A preferred reducing agent is DTT. The concentrationof DTT is preferably in the range from 1 to 30 mM, more preferably theconcentration of DTT is in the range from 5 to 15 mM, in particularabout 1 mM. The chelating agent is preferably EDTA. The concentration ofEDTA is preferably in the range from 1 to 15 mM, more preferably theconcentration of is in the range from 2 to 10 mM, in particular about 5mM. Suitable incubation conditions are for example a temperature in therange from 20 to 30° C., preferably about 25° C. a duration of 20 min to2 h, preferably about 1 h and a shaking speed in the range from 200 to2000 rpm preferably about 1000 rpm.

Under these conditions the VLP disassembles into VP1 pentamers. The VP1pentamers can for example be reassembled into icosahedral VLPs bydialysis against 2×2 liter reassembly buffer (10 mM TrisHCl pH 7.4; 150mM NaCl; 2 mM CaCl₂) for at least 84 h. In order to produce a VLP withthe fusion protein the fusion protein may be incubated with the VP1pentamers during the disassembly procedure (see above). The fusionprotein and VP1 pentamers than reassemble during dialysis into mixedVLPs.

According to one embodiment of the process further comprises the steps:

-   f) disassembly of the VLP into pentamers;-   g) mixture of the pentamers with wildtype VP1 pentamers; and-   h) reassembly of VLPs from the pentamer mixture.

An overview of the process is shown in FIG. 2.

The fusion protein according to the first aspect can for example beincorporated into the capsid envelope by co-expression of the respectivefusion protein and VP1 in a suitable host cell e.g. a eukaryotic cell.In a preferred embodiment of the invention the fusion protein isco-expressed with a VP1 or a fusion protein comprising the VP1. Inparticular the fusion protein and the VP1 are coexpressed in Sf9 cells.

Active substances can be incorporated into the interior of the capsidenvelope by for example dissociation of the capsid envelope andsubsequent re-association in the presence of the active substance or byosmotic shock of the VLP in the presence of the active substance.

EXAMPLES Example 1: Cloning of VLP-Variants with a Cyclic Lyp-1-Peptidein Via Whole Vector PCR

a) Introduction of the cyclic Lyp-1-peptide into the HI-loop

Primers:

Lyp-1peptid_HI-Loop FW (SEQ ID NO: 40)GAACTAGAGGGTGCGGATCCGAAGAGGAAGAGGGTTCCCAGCAGTGG Lyp-1peptid_HI-Loop RV(SEQ ID NO: 41) GCTTGTTACCACAACCTGATCTACGTCTACGGTTGGTGAACATGCPlasmid:pTXB1_VP1 (SEQ ID NO: 42)

Before the PCR the primers were phosphorylated 20 min at 37° C. and 10min at 75° C. in the following reaction set up:

50 pmol primer 2 μl buffer A 2 μl 10 mM dATP 10 U T4 polynucleotidekinase (Fermentas) ad 20 μl bidest H₂O.

The following PCR mixture was prepared:

1.00 μl pTXB1_VP1 (1 ng/μl) 4.00 μl 5x PCR Q5 Puffer (w 7.5 mM MgCl2)0.40 μl 10 mM dNTP (200 μM) 1.00 μl Lyp-1peptid_HI-Loop_FW (10 μM) 1.00μl Lyp-1peptid_HI-Loop_RV (10 μM) 0.20 μl Q5-Hot Start (2 U/μl NEB) (0.4U) 12.40 μl  H₂O MilliQ

For PCR, the following temperature profile was used: An initialactivation at 98° C. for 30 sec followed by 25 repetitions of thefollowing cycle steps 1 to 3: 1) Denaturation: 98° C. for 10 seconds; 2)Annealing: 60° C. for 10 seconds; and 3) Extension: 72° C. for 3 min.After the temperature cycling, the samples were again kept at 72° C. for10 min and finally cooled to 12° C. until the samples were retrieved.

PCR products (7771 bp) were separated by agarose gel electrophoresis andfragments of about 7400 bp were eluted using the QiaEx Kit (Qiagen).

Fragments were eluted with 30 μl of 70° C. buffer 4 (NEB) andsubsequently incubated with 2 μl DpnI (digest of methylated templateplasmid DNA) for 2 h at 37° C. followed by an additional 20 min at 80°C. for inactivation of the DpnI. Afterwards the PCR fragments werereligated to using the T4 ligase.

Ligation Reaction Set Up:

2 μl PCR-Product 1 μl 10x Ligase-buffer 2.5 Units Ligase Ad 10 μl ddH₂O

For ligation the samples were incubated for 1 h RT.

The plasmids were then transformed into competent DH5α bacteria bythermal shock. Accordingly, 5 μl ligation sample were added to 50 μlcompetent XL1Blue cells and incubated for 30 min on ice, heated for 45 sat 42° C. followed by further incubation for 2 min on ice. Afterwards,500 μl SOC were added and the cells were incubated for 30 min at 37° C.The cells were then harvested for 5 min at 3.000×g, the cell pelletsresuspended in 50 μl SOC and 20 μl of the cell suspension spread on“amp+” agar plates.

Single colonies growing on the amp+ agar plate were tested for correctinsertion. For this, the plasmid DNA was prepared by DNA Miniprep(Qiagen) according to the manufacturers' protocol. The obtained DNA wasdigested with the restriction enzyme Agel. A construct with a correctinsertion would yield two bands in agarose gel electrophoresis at 6703base pairs and 1068 base pairs. Clones with correct restriction patternwere sequenced.

b) Introduction of the Cyclic Lyp-1-Peptide into the DE-Loop

For introduction of the Lyp-1-peptide into the DE-loop the protocol asdescribed under a) was used with the following differences.

Primers:

Lyp-1peptid_DE-Loop_FW (SEQ ID NO: 43)GAACTAGAGGGTGCGGATCCGAAGAGGAAGAGGGTGCTGGCAAGC Lyp-1peptid_DE-Loop_RV(SEQ ID NO: 44) GCTTGTTACCACAACCTGATCTACGTCTACGGTGGGTAGCCTGGC

The expected band size in the restriction analysis was 6760 base pairsand 1011 base pairs DE-Loop.

c) Introduction of the Cyclic RGD-Peptide into the HI-Loop

For introduction of the RGD-peptide into the HI-loop the protocol asdescribed under a) was used with the following differences.

Primers:

cRGDpeptid_VP1-HI-Loop_FW  (SEQ ID NO: 78) GATTCATGCGGATCCGAAGAGGAcRGDpeptid_VP1-HI-Loop_RV  (SEQ ID NO: 79) ACCTCTACCACAACCTGATCTACGTCTAC

The plasmid template was pTXB1_VP1-HI-Loop-Lyp1, which is the resultingpeptide from the protocol described under a) including the introducedLyp1 peptide.

The PCR product had a size of 7765 bp.

The obtained DNA was digested with the restriction enzyme BamHI. Aconstruct with a correct insertion would yield two bands in agarose gelelectrophoresis at 6695 base pairs and 1070 base pairs. Clones withcorrect restriction pattern were sequenced.

Example 2: Expression and Purification of VP1-Lyp1 Fusion Proteins in E.coli

a) Protein Expression

Expression vectors pTXB1_VP1-HI-Loop+Lyp1 and pTXB1_VP1-DE Loop+Lyp1were produced according to Example 1.

For expression in E. coli the plasmids were transformed into the E. colistrain BL21 (DE3). For transformation, chemically competent cells weremixed with 1 ng of plasmids and incubated on ice for 30 min.Transformation was induced by heat shock for 45 s at 42° C. After thiscells were transferred back to ice for at least 2 min before addition ofSOC medium (37° C.) and incubation for 1 h at 37° C. under continuousshaking (225 rpm). For selection of positively transformed E. coli BL21the culture was spread out on LB agar plates containing ampicillin(LB-amp) as selective marker. For expression, a colony grown on theLB-amp plate was inoculated into 50 ml LB medium comprising ampicillinin a concentration of 100 μg/ml and grown overnight at 37° C. and 225rpm. This overnight culture was transferred into one liter LB-ampmedium. Inoculated cultural medium was split into four 250 ml fractionsand transferred into 1000 ml Erlenmeyer flasks. The four cultures weregrown at a temperature of 37° C. with a shaking speed of 225 rpm untilreaching an OD600 of 0.4. At this growth stage, protein expression wasinduced by an addition of 0.4 mM IPTG (end concentration). Afterinduction of protein expression, the cells were grown for four hours at30° C. with 225 rpm shaking.

b) Protein Preparation

Cells were harvested by centrifugation at 3,500 g and 4° C. for 15minutes. The supernatant was discarded and the pellets were resuspendedin 15 ml lysis buffer, joining two resuspended pellets in one 50 mlfalcon tube.

Lysis Buffer

20 mM Tris-HCl, pH 8.5

500 mM NaCl

1 mM EDTA

1 mM TCEP

0.1% Triton-X 100

Lysozyme was added at a concentration of 20,000 units per milliliter ofcell suspension. The cell suspension was then incubated for 30 minutesin an overhead shaker at 4° C. After incubation into each of thefalcons, 5 g of silica beads (0.1-0.2 mm, Mühlmeyer) were added. Thecells were lysed in a BigPrep adapter with the FastPrep instrument(MPBio) at 6.5 m/s for 30 seconds in two repetitions. The lysed cellswere then centrifuged at 10,000 g and 4° C. for 15 minutes. Thesupernatants were recovered and stored for further processing. Thepellets were then resuspended in 15 ml Lysis Buffer and lysed using theFastPrep system as described above. The cells were again centrifuged at10,000 g and 4° C. for 15 minutes. The supernatants were retrieved andjoined with the stored supernatants.

c) Protein Purification

10 ml of chitin agarose were filled into a column. The column was washedwith 100 ml of Column Buffer and stored at 4° C.

Column Buffer

20 mM TrisHCL, pH 8.5

500 mM NaCl

1 mM EDTA

The supernatants of the protein preparation were again spun at 15,000 gat 4° C. for 30 minutes and afterwards applied onto the chitin column.The protein solution was allowed to pass the chitin column by gravityflow. After the protein solution has passed the column by gravity flow,the chitin column was washed with 200 ml column buffer by gravity flow.After the column buffer has run through, 30 ml cleavage buffer wereadded.

Cleavage Buffer

20 mM TrisHCl, pH 8.5

200 mM NaCl

1 mM EDTA

15 mM DTT

After the Cleavage Buffer has entered the gel, the chitin column wasclosed and incubated overnight at room temperature. After incubation,the cleavage buffer was removed by gravity flow. For elution, five 10 mlportions of elution buffer were added and let run through the columncollecting the eluate.

Elution Buffer

20 mM TrisHCl, pH 8.5

200 mM NaCl

1 mM EDTA

2 mM DTT.

During the preparation and purification, samples were collected at thedifferent steps and tested together with samples of the eluate usingSDS-PAGE and Western

d) Analysis of the Protein Production

SDS-PAGE was performed with two gels containing the same samples. Onegel was stained with InstantBlue. The second one was used for WesternBlot analysis. The proteins were transferred from the gel to a membranefor one hour at 1 mA/cm². Afterwards, free protein binding sites on themembrane were blocked for 30 minutes at room temperature in PBS-Tcontaining 5% (w/w) low fat milk powder. The low fat milk solution wasremoved and the first antibody AK 254 C7E4 diluted 1 to 5,000 in PBS-Tplus 0.5% (w/w) low fat milk powder was added to the membrane andincubated for one hour at room temperature. After incubation, themembrane was incubated three times for five minutes in PBS-T. The PBS-Twas removed and the second antibody was applied. For this, a goatanti-mouse POX was diluted 1 to 10,000 in PBS-T+0.5% low fat milkpowder. The antibody solution was added to the membrane and incubatedfor one hour at room temperature. The membrane was again washed threetimes for 5 minutes in PBS-T. Bound secondary antibody was detected bychemoluminescence. Eluate fractions showing a positive signal werepooled and the volume was reduced by a factor of 10 using Viva spin 20columns (molecular weight cut off 30,000). Protein content and purity ofthe final eluate were determined via an Experion analysis system(Biorad).

Example 3: Expression of VP1 in Sf9 and Production of VLP

a) Protein Expression

Sf9 were grown to a cell density of 2×10⁷ in serum-free TC100 medium andinfected with the recombinant baculovirus with a multiplicity ofinfection (MOI) of 5. After infection the cells were grown for 5 to 7days at 27° C. producing the VP1 protein encoded by the baculovirus. Theproduced protein is secreted into the expression medium. In the cellsupernatant the secreted proteins self-assemble into VLPs.

b) Purification of VLPs from the Supernatant

Sf9 cells were separated from the supernatant by centrifugation for 5min at 500×g. Cells were discarded and the supernatant was centrifuged asecond time for 90 min at 5000×g in order to remove larger impurities.

The VLPs were then separated by ultracentrifugation. For this, 15 ml ofclarified supernatant was loaded on 3 ml 40% sucrose.

Ultracentrifugation was performed in a Sorwall MX150 for 4 h at 100000×gand 4° C. In the centrifugation tube a pellet formed by the VLPs whichwas harvested. The VLP containing pellet was resuspended in Tris buffer(10 mM Tris, 150 mM NaCl, pH 7.5) The protein concentration of theresuspended VLPs was determined and adjusted to 0.5 μg/μl by theaddition of Tris-Buffer.

Example 4: Production of VLPs Containing a VP1-Lyp1 Fusion Protein

FIG. 2 gives an overview of the production. In a first step, VP1 wasproduced as described in Example 3. VP1 with a Lyp-1 targeting peptidein the HI or DE loop were produced as described in Example 2.

The proteins were then mixed in a ratio of 5:1. Accordingly, 100 μl VP1(500 ng/μl) were added to 200 μl VP1-HI-Loop-Lyp1 from E. coli (at aconcentration of 50 ng/μl). The mixed VLPs were then disassembled byaddition of 3 μl 0.5 M EDTA and 3 μl 1 M DTT at an incubation of 1 hourat 25° C. and 1,000 rpm shaking. The reassembly by dialysis against acalcium rich buffer then leads to the formation of mixed VP1 VLPs.

The sample was dialysed for 60 hours against 2 liters of ReassemblyBuffer.

Reassembly Buffer

PBS

1 mM CaCl₂

1 mM MgCl₂

After 60 hours, the Reassembly Buffer was replaced by another 2 litersof the same buffer and the sample was dialysed for another 24 hours.Afterwards, the protein concentration in the sample was determined usingthe PIERCE 660 nM protein assay. Samples were analysed using SDS-PAGEand Western Blot Analysis. According to the same protocol VLPscontaining VP1-DE-Loop-Lyp1 were produced.

Example 5: Targeting Test with VLPs Containing a VP1-Lyp1 Fusion Protein

a) Labelling of VP1 with Fluorescent Dyes

VP1 was produced using the established SF9/Baculo virus expression.

200 μl VP1 (0.5 mg/ml) from insect expression system were mixed with 10μl of 0.2 M bicarbonate pH 9 and 0.5 μl 10 mM NHS-Ester of thefluorescent dye. The mixture was rotated at RT for 1 h before 1 μmol ofethanolamine was added to quench unreacted dye. Free dye was removed bydialysis against 2×1 Liter PBS containing 1 mM CaCl₂ and 1 mM MgCl₂ 24 heach.

b) Production of VLPs with VP1-Lyp1 Fusion Proteins and Dye Labelled VP1

The following VLPs assembled by VP1-Lyp1 fusion proteins and dyelabelled VP1 were produced according to the protocol in Example 3.

-   -   i) VLP with VP1-DE-Loop-Lyp1 and VP1-FITC    -   ii) VLP with VP1-HI-Loop-Lyp1 and VP1-FITC    -   iii) VLP with VP1-DE-Loop-Lyp1 and VP1-Atto488    -   iv) VLP with VP1-HI-Loop-Lyp1 and VP1-Atto488    -   v) VLP with VP1-DE-Loop-Lyp1 and VP1-Atto647    -   vi) VLP with VP1-HI-Loop-Lyp1 and VP1-Atto647        c) Treatment of Cancer Cells Exhibiting p32 with the VLPs

The cancer cell line MDA-MB-231 (ATCC® HTB-26™) was applied into a12-well plate with a cell number of about 200.000 cells per well. Thecells are incubated at 37° C. and 5% CO₂ in 2 ml of the culture mediumDMEM. After 20 h of incubation 4 μg of a VLP construct as defined in i)to vi) was added in the culture medium and the cells were incubated foranother 4 h at 37° C. and 5% CO₂.

d) Flow Cytometry

After incubation with the VLP the culture medium was removed and thecells were washed by addition and subsequent removal of 2 ml of ice coldPBS. In the following cells were dissociated from the surface of thebottom of the well by addition of 500 μl T/E solution. To the cells 2 mlof PBS+2% (w/w) fetal calve serum (FCS) were added and the cellsuspension were centrifuged for 10 min at 250 g and 5° C. Afterwards thecells were washed three times with 2 ml of PBS+2% (w/w) FCS. The cellsuspension were centrifuged for 10 min at 250 g and 4° C. and theresulting cell pellet suspended in 1 ml PBS with 1 μg/ml propidiumiodide.

The results of the flow cytometry are shown in form of histograms inFIG. 3 a) to c). FIG. 3 shows histograms calculated from flow cytometrydata obtained from MDA-MB-231 cells treated with VLPs. The histogramcurves in FIG. 3 a) to c) correspond to treatments with the VLPs fromVP1, VP1-DE-Loop-Lyp1 or VP1-HI-Loop-Lyp1. In addition, a histogram froma control sample of untreated cells is present. In the histograms theX-axis represents the intensity of the measured signal, the Y-axis thenumber of cells with the specific signal intensity. In all experimentsthe histograms of the cell treatment with VLPs containingVP1-DE-Loop-Lyp1 or VP1-HI-Loop-Lyp1 are shifted to the right ascompared to the cell treatment with VLPs containing only wildtype VP1.Thus, a larger number of cells take up the VLPs containing VP1 proteinswith the targeting peptide (Lyo-1 as targeting region) according to theinvention. An explanation for the increased uptake is the interaction ofthe Lyp-1 targeting region with the cancer cells exhibiting p32.

The results of the experiment are additionally shown in FIG. 4 in formof a column diagram. In FIG. 4 each column represents the percentage ofcells with a positive uptake of VLPs, identified by minimum signalstrength. FIG. 4 shows that the number of cells with a positive uptakeof VLPs is increased by about 20% in the experiments with VLPscontaining VP1-DE-Loop-Lyp1 and VP1-HI-Loop-Lyp1 compared to VLPs fromnormal VP1.

Example 6: Clinically Relevant Breast Cancer Spheroid Model

a) Cells

Three breast cancer cell lines purchased from ATCC were selected. Thesecell lines represent the most relevant breast cancer cohorts, are knownto form compact spheroids and incorporate the Atto-488 labeled VLPs.Namely, SKBr-3, passage 12 represents Her2/neu positive breast cancers,MCF-7, passage 11 the hormone receptor positive tumors and Hs578T,passage 17 the triple negative carcinomas.

For the preparation of heterotypic breast cancer spheroids, organspecific breast cancer fibroblasts in an early passage (F4616a, passage4) and PBMC from a healthy donor were used. The breast cancer cell lineswere cultured according to the recommendations published by ATCC. Theprimary fibroblasts were cultured in DMEM/F12 medium substituted with10% FCS. The PBMC were isolated from lithium heparinized whole bloodaccording to standard procedure and freshly used for spheroidpreparation.

b) Preparation of Heterotypic Breast Cancer Spheroids

For spheroid formation single cell suspension was obtained bytrypsinization of the monolayer cultures. Cell vitality was determinedby trypan blue exclusion test: SKBr-3 cells: 92.4%, MCF-7 cells: 94.8%,Hs578T cells: 93%, primary breast cancer fibroblasts: 96.6% and PBMC:99.6%.

For the preparation of heterotypic breast cancer spheroids single cellsuspensions were mixed in a ratio of cancer cells (2): fibroblasts (1):PBMC (1). In each 96-well 50.000 mixed suspension cells were given in avolume of 100 μl DMEM/F12 based cell culture medium. Compact spheroidswere generated according to SpheroTec's 3D technology for 48 h.

c) VLP Samples and Incubation of the Heterotypic Breast Cancer Spheroids

Three different types of VLPs labelled with Atto-488 were used asfollows: VP1, VP1-HI-Loop-linRGD (an epitope of three repetitive RGDmotifs), and VP1-HI-Loop-cycRGD (a cyclic RGD motif as described above,i. e. the RGD motif is comprised in the targeting region of the fusionprotein according to a first aspect of the invention). The heterotypicbreast cancer spheroids were incubated with 20 μl from each VLP and themock control (supernatant from untransfected host cells) for six hours.All incubations were performed in triplicates.

After 6 h incubation with the VLP samples the heterotypic breast cancerspheroids were fixed in 10% PBS buffered formalin. After dehydration thespheroids were embedded in paraffin blocks according to a standardpathologic protocol.

d) Detection of the Bio-Distribution of the VLP Samples

For fluorescence microscopy 3 μm sections were prepared from theparaffin blocks. After deparaffinization and rehydration of the spheroidsections, bio-distribution of the Atto-488 labelled VLP samples wasdocumented photographically using fluorescence microscopy.

All tested VLP samples (see above) penetrated into the compactheterotypic breast cancer spheroid models within six (6) hoursincubation time and showed an intracellular accumulation. Exemplarily,results are shown for the breast cancer cell line SKBr3. Thearchitecture of the SKBr-3 spheroids was organized of aggregated tumornodules. These tumor nodules mainly consisted of central fibroblastssurrounded by cancer cells. Leukocytes were found in both compartments.FIG. 5 shows the results of the breast cancer cell line SKBr3. Additionof the RGD motif leads to an increased VLP accumulation in the spheroid.The cyclic variant (VP1-HI-Loop-cycRGD) is much more effective than thelinear one (VP1-HI-Loop-linRGD) demonstrating the potential of thestrategy of cyclic peptide addition to VP1, i. e. of an exemplary fusionprotein according to the present invention.

Example 7: Orthotopic Breast Cancer Tumor Model

a) Study Design (See Also FIG. 6)

The study consisted of 2 experimental groups, each containing either 3(animal control) or 9 (VP1-HI-Loop-Lyp1) female BALB/c nude mice afterrandomization. On day 0, 5×10⁶ MDA-MB-231-Luc-Z1 tumor cells in 100 μlPBS:Matrigel were orthotopically implanted into 12 female BALB/c nudemice.

In the following, the growth of the orthotopically implanted primarytumors were determined twice weekly by caliper measurement. On day 20,tumor-bearing animals were randomized according to measured primarytumor volumes. On the following day (day 21), animals were once treatedi.v. with 45 μg/animal (150 μl) of VP1-HI-Loop-Lyp1 or salt solution(animal control), respectively.

On days 21/22, 4 h, 13 h and finally 24 h after a single treatment, therespective animals were sacrificed and a necropsy performed.

At necropsy, animals were killed by cervical dislocation. Primary tumorswere collected and wet weights and volumes determined. Primary tumortissues were divided into two parts. One part was snap-frozen in liquidnitrogen and stored appropriately at −80° C., whereas the other part wasanalyzed by flow cytometry.

b) Flow Cytometry

One half of the primary tumors were chopped with a scalpel and digested45-60 min at 37° C. for cell isolation (2 mg/ml collagenase (0.21 U/ml)and 25 μg/ml DNAse in PBS). Thereafter red blood cells were lysed andthe remaining isolated cells stained with an anti-human CD46-APCantibody (BD Pharmingen, #564253). Flow cytometry analysis was performedusing a BD-ExCalibur FACS analyzer. For each time point, an isotypecontrol staining (mouse IgG2a, k-APC; BD-Pharmingen; #555576) wasperformed for one tumor sample.

FIG. 7 demonstrates uptake of fluorescently labelled VLP-HI-Loop-Lyp1 inthe tumors at each time point. At early time points uptake was morepronounced, pointing to a very fast kinetic. No fluorescence was foundin mouse cells surrounding and penetrating the tumor.

Thus, targeting to tumor cells in vivo of VLP comprising a fusionprotein according to a first aspect of the present invention has beendemonstrated.

The invention claimed is:
 1. A fusion protein comprising at least afirst and a second peptide, wherein the second peptide comprises atargeting region and a first and a second interaction region, the firstand the second interaction region form a stem and the targeting regionforms a loop, the second peptide is located on the surface of the fusionprotein, the second peptide comprises at least two interaction pairs,wherein an interaction pair is formed by an amino acid of the firstinteraction region and an amino acid of the second interaction region,at least one interaction pair is a covalent interaction pair in whichthe amino acids are covalently bound, and the first peptide is VP1 fromJVC.
 2. The fusion protein according to claim 1, wherein the covalentinteraction pair is formed by a cysteine in the first interaction regionand a cysteine in the second interaction region.
 3. The fusion proteinaccording to claim 1, wherein the amino acid sequence of the targetingregion is selected from the group consisting of Lyp-1 (SEQ ID NO: 1),RGD (SEQ ID NO: 60), RGR, HER-2 binding peptide (SEQ ID NO: 2), CREKApeptide (SEQ ID NO: 3), NGR peptide CPP-2 (SEQ ID NO: 4), CPP-44 (SEQ IDNO: 5), F3 (SEQ ID NO: 6), RMS-P3 (SEQ ID NO: 7), F56 (SEQ ID NO: 8),LTVSPWY-peptide (SEQ ID NO: 9), and WNLPWYYSVSPT-peptide (SEQ ID NO:10).
 4. A virus-like particle (VLP) comprising at least one fusionprotein according to claim
 1. 5. The VLP according to claim 4, whereinthe second peptide is located on the outer surface of the VLP capsid. 6.The VLP according to claim 4, further comprising a second fusion proteincomprising a VLP binding protein and an exogenous peptide.
 7. The VLPaccording to claim 4, wherein the VLP comprises no cargo.
 8. The VLPaccording to claim 4, wherein the VLP comprises a cargo selected fromsingle-stranded or double-stranded DNA or RNA, preferably siRNA,oligopeptides, polypeptides, hormones, lipids, carbohydrates, othersmall organic compounds or mixtures thereof.
 9. A method of treating asubject having a disease or diagnosing in a subject a disease, themethod comprising administering the VLP according to claim
 4. 10. A drugdelivery system comprising the VLP according to claim
 4. 11. Apharmaceutical composition comprising the VLP according to claim 4 andat least one pharmaceutically acceptable carrier.
 12. An isolatedpolynucleotide comprising a nucleic acid sequence encoding a fusionprotein according to any claims 1 to
 3. 13. An expression vectorcomprising the polynucleotide according to claim
 12. 14. A host cellcomprising the expression vector according to claim
 13. 15. A process ofproducing the VLP according to claim 4, the process comprising the stepsof: a) introducing an isolated polynucleotide comprising a nucleic acidencoding a fusion protein according to claim 1 into a host cell; b)culturing the transformed host cell in a medium under conditions leadingto a protein expression with the nucleic acid as a template; c)isolating the expression product from the cell; and d) assembly of theexpression product optionally with further viral proteins into the VLP.16. The process according to claim 15, further comprising the steps: e)disassembly of the VLP into pentamers; f) mixture of the pentamers withwildtype VP1 pentamers; and g) reassembly of VLPs from the pentamermixture.